The role of intracellular chloride in hyperpolarizing post-synaptic inhibition of crayfish stretch receptor neurones

Abstract

1. The intracellular Cl(-) activity (a(Cl) (i)) of isolated crayfish stretch receptor neurones was measured using liquid ion exchanger Cl(-)-selective micro-electrodes. The potential developed due to the difference between the normal extracellular Cl(-) activity (a(Cl) (o)) and a(Cl) (i) (V(Cl)) was compared with the simultaneously measured reversal potential of the inhibitory post-synaptic potential (E(i.p.s.p.)) to further clarify the ionic basis of the i.p.s.p..2. In normal Ringer solution, V(Cl) (63.3 +/- 2.3 mV) was found to be close to the resting membrane potential (E(m), 62.6 +/- 3.9 mV) while E(i.p.s.p.) (74.5 +/- 1.9 mV) was more negative than either. The V(Cl) value corresponds to an apparent a(Cl) (i) of 12.7 +/- 1.3 mM, which is about 4 mM more than required for a Cl(-) governed E(i.p.s.p.) of 74.5 mV.3. Reducing a(Cl) (o) caused smaller changes in V(Cl) than predicted for passive Cl(-) re-distributions. On complete removal of extracellular Cl(-) (Cl(o) (-)), V(Cl) increased to 84.6 +/- 2.7 mV, equivalent to an apparent a(Cl) (i) of about 5 mM-Cl(-). This value can be used as an estimate of the level of intracellular interference on the Cl(-)-selective micro-electrode.4. Increasing extracellular K(+) (K(0) (+)) decreased both V(Cl) and E(i.p.s.p.). Decreasing K(o) (+) had the converse effect. The time course of the changes in V(Cl) and E(i.p.s.p.) was much the same. The difference between V(Cl) and E(i.p.s.p.) decreased to about 3 mV in high K(o) (+), and increased to about 30 mV in low K(o) (+). This variation in the difference between E(i.p.s.p.) and V(Cl) is consistent with the assumption that anions other than Cl(-) contribute to the recorded V(Cl) rather than another ion contributes to the inhibitory current.5. Application of 5 mM-NH(4) (+) or of frusemide (6 x 10(-4) M) decreased V(Cl) and E(i.p.s.p.). The difference between V(Cl) and E(i.p.s.p.) was also decreased.6. We conclude that a(Cl) (i) is lower than predicted from a passive distribution and thus the chloride equilibrium potential (E(Cl)) is more negative than E(m). If a constant intracellular interference equivalent to about 4 mM-Cl(-) is assumed to contribute to the recorded V(Cl), E(Cl) was approximately equal to E(i.p.s.p.) in all the experimental conditions. Therefore we suggest that the i.p.s.p. is solely generated by Cl(-) ions.